Protein synthesis in Rous sarcoma virus-transformed cultured chick embryo fibroblasts (CEF) has been reported to be more sensitive to inhibition by diphtheria toxin (DT) than protein synthesis in normal CEF. Here we have examined the DT sensitivity of CEF infected with Schmidt-Ruppin Rous sarcoma virus (SR-RSV), its transformation defective mutant ts 68, avian myeloblastosis virus (AMV) and polyoma transformed BHK-21 cells to see if the increased sensitivity to DT correlated with in vitro cell transformation and/or with virus production. Protein synthesis in SR-RSV transformed cells was 10 times more sensitive to DT than protein synthesis in normal CEF. Similarly, BHK-21 cells transformed with polyoma virus were 7 to 8 times more sensitive to DT than primary BHK or a continuous culture of BHK-21 cells, even though no virus was produced. Furthermore, CEF infected with SR-RSV ts 68, a temperature sensitive transformation defective mutant, were more sensitive to DT at the permissive temperature than at the nonpermissive temperature, even though virus is produced at both temperatures. In addition, CEF infected with AMV, a non-transforming virus, showed no increase in sensitivity to DT. It is proposed that increased sensitivity to DT is a newly discovered property of at least some virus transformed cells and is independent of virus production.